Allosteric inhibition of TEM‐1 β lactamase: Microsecond molecular dynamics simulations provide mechanistic insights

Abstract β‐lactam antibiotics target DD‐transpeptidases, enzymes that perform the last step of bacterial cell‐wall synthesis. To block the antimicrobial activity of these antibiotics, bacteria have evolved lactamases that render them inert. Among these, TEM‐1, a class A lactamase, has been extensively studied. In 2004, Horn et al. described a novel allosteric TEM‐1 inhibitor, FTA, that binds distant from the TEM‐1 orthosteric (penicillin‐binding) pocket. TEM‐1 has subsequently become a model for the study of allostery. In the present work, we perform molecular dynamics simulations of FTA‐bound and FTA‐absent TEM‐1, totaling ~3 μS, that provide new insight into TEM‐1 inhibition. In one of the simulations, bound FTA assumed a conformation different than that observed crystallographically. We provide evidence that the alternate pose is physiologically plausible and describe how it impacts our understanding of TEM‐1 allostery.

The main text describes MD simulations of apo and holo (FTA-bound) TEM-1. The sequences of these two proteins are identical and correspond to entry S#864 in the Lactamase Engineering Database (LacED) 1 (i.e., the 1ZG4 crystal-structure sequence). We also simulated a third holo (FTA-bound) system whose sequence corresponded to LacED entry S#256 (i.e., the 1PZP crystal-structure sequence). The S#864 and S#256 sequences differ by only three amino acids, so the two holo simulations behaved similarly. To simplify the discussion and presentation of results, we described only the S#864 holo simulation in the main text. Here we additionally describe the S#256 holo simulation, which was generally similar to the S#864 holo simulation except it did not capture the horizontal FTA pose. For clarity's sake, we will refer to S#864 TEM-1, described in the main text, as wildtype (WT) TEM-1. We will refer to S#256 TEM-1 as mutant TEM-1.

Simulation equilibration
To determine how much the TEM-1 conformations changed over the course of the simulations, we aligned them and calculated the backbone-atom root-meansquare distances (RMSD) between each frame and the respective initial frame ( Figure S1). The WT apo, WT holo, and mutant holo TEM-1 simulations are shown on the top, middle, and bottom rows, respectively.
We discarded the first 10 ns of each simulation (marked with a dashed vertical line) to ensure all simulations had properly equilibrated.
The WT apo simulations deviated more from the starting position (〈 !"# 〉 = 1.15 ± 0.17 Å) than the WT holo simulations (〈 $#%# '( 〉 = 0.91 ± 0.10 Å), though the deviations were modest in both cases. These findings harmonize with previous nuclear magnetic resonance (NMR) 2 and molecular dynamics (MD) studies [3][4][5] , which have shown that TEM-1 is a highly rigid protein with high order parameters (〈 ) 〉 = 0.90 ± 0.02) and only minor conformational changes in simulations (per RMSD). Figure S1. Plots of the TEM-1 backbone RMSD to the first frame. In the top row, the WT apo (S#864) simulations. In the middle row, the WT holo (S#864) simulations. In the third row, the mutant holo (S#256) simulations. The two graphs on each row are identical, except the second graph uses a logarithmic scale on the X axis.

FTA decreases TEM-1 flexibility
We calculated differences in per-residue RMSF values, apo -holo, for the mutant holo simulations, just as we did for the WT holo simulations ( Figure S2). With few exceptions, the WT and mutant holo simulations differed similarly from the apo simulation. Figure S2. Differences in center-of-geometry RMSF per residue. RMSF differences between the two holo simulations are small (less than 0.3 Å), except for the coil after α7, where holo mutant residues are more flexible than holo WT residues. There is one outlier according to COG RMSF: residue 100. This residue is one of the mutations observed in 1PZP (N100R), but if we consider the RMSF of only Cα atoms, the holo simulatios differ by only 0.026 Å. Catalytic residue S70 and the general base E166 have lower RMSF values for the holo mutant simulations, but the changes fall below 0.3 Å.

FTA impact on selected residues
In the main text, we provide Janin plots for Y105 and R244, WT apo vs. WT holo (Figure 4 A and 4B). We did not include Janin plots for other residues implicated in ligand recognition, ligand stabilization, and catalysis because FTA binding did not have a substantial impact on their side-chain dynamics. But we include these plots here for completeness' sake, together with Janin plots for the mutant holo simulations ( Figure S3).   Additionally, residues at the β4 N-terminus have higher BC values in the holo simulations than in the apo simulations. This includes R244, which tends to be further from the catalytic pocket in the holo simulations. We note that large BC differences in the α11 and α12 helices (A217-A224 and M272-H289) are likely artefactual; when FTA is present (holo), the gap it forms decreases the BC for some residues in these helixes.